Complications Infections & Malignancies (Videos Available)

Wednesday July 04, 2018 from 09:45 to 10:45

Room: N-117/118

527.5 Delineation of the viral and host cell genomic alterations in EBV-positive PTLD

Award Winner

Yarl Balachandran, United States has been granted the TTS Young Investigator Scientific Award

Yarl Balachandran, United States

Surgical Resident, Postdoctoral Scholar in Transplantation Surgery
Stanford University Medical Center


Delineation of the Viral and Host Cell Genomic Alterations in EBV-positive PTLD

Yarl Balachandran1, Marla McPherson1, Scott D. Boyd2, Carlos O. Esquivel1, Sheri Krams1, Olivia M. Martinez1.

1Transplant Immunology Laboratory/Division of Abdominal Transplant, Stanford University, Stanford, CA, United States; 2Department of Pathology, Stanford University, Stanford, CA, United States

Post-transplant lymphoproliferative disorder (PTLD) is a potentially fatal complication of organ transplantation characterized by abnormal proliferation of lymphoid cells in the setting of immunosuppression after transplantation. Epstein-Barr virus (EBV) is responsible for abnormal lymphocyte proliferation in 50–80% of PTLDs, particularly in early-onset disease. Latent membrane protein 1 (LMP1) is the chief oncogenic protein of EBV and has been shown to be critical in the transformation of human B cells by the virus. LMP1 may activate several cellular signal transduction pathways including MAPK/ERK, p38/JNK, PI3K/AKT and NF-kB/AP-1. The ability to usurp and dysregulate signaling pathways may explain how LMP1 promotes cell transformation, survival, and proliferation. Our laboratory has shown that LMP1 isolated from EBV-associated B cell lymphoma lines of PTLD patients contains gain-of-function mutations at AA212 (G-S) and AA366 (S-T) that result in sustained ERK signaling, c-Fos activation, and AP-1 activity. In this study, we asked whether these mutations, or other genetic alterations, are present in primary EBV+ PTLD tumors themselves.
DNA was isolated from formalin-fixed paraffin-embedded tissue sections of EBV+ PTLD tumors (n=8). Nested PCR was used to amplify LMP1, and the PCR products generated were cloned and sequenced. The presence or absence of gain-of-function mutations at AA212 (G-S) and AA366 (S-T) were assessed. 7 out of the 8 EBV+ PTLD tumors demonstrated both gain-of-function mutations in LMP1. Furthermore, 6 out of the 8 tumors contained an extra repeat of 8 amino acids within the LMP1 signaling tail corresponding to a putative JAK3 binding motif. We have previously identified this repeat in 3 of 6 EBV+ B cell lymphoma lines from PTLD patients. LMP1 was also cloned from the whole blood of a pediatric small bowel PTLD patient. In addition to these 2 mutations being present, the 8 amino acid repeat was also present but in triplicate, suggesting this motif may be crucial to the oncogenic activity of LMP1. In order to assess host cell mutations in EBV+ PTLD tumors, a qBiomarker Mutation PCR array was performed for the PI3K/AKT/mTOR pathway, known to be important in human malignancies. While mutations in PI3K/AKT/mTOR were identified in EBV+ B cell lymphoma lines from PTLD patients, there were significantly more mutations in the primary EBV+ PTLD tumors, with 25 distinct mutations found within the PTEN, PI3K, and STK11 molecules. 3 distinct mutations were identified in more than 1 tumor, and 4 distinct mutations were shared between a cell line and tumor.
Our findings clearly demonstrate that key gain-of-functions mutations in LMP1 detected in blood and cell lines are also detected in the primary tumor, suggesting a role in tumorigenesis and great potential as biomarkers of EBV+ PTLD. Moreover, host cell mutations may also contribute to dysregulation of key signal transduction pathways in EBV+ PTLD.

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